Abstract:

In order to construct Placenta special gene 8 (PLAC8) knockout human embryonic kidney cell line (293T) and detect the effect of PLAC8 knockout on cell proliferation, based on CRISPR/Cas9 and flow cytometry technology, a PLAC8 knockout 293T cell line was constructed. Edited cells were screened by genome sequencing and T7E1 enzyme digestion. The total protein of the selected cells was extracted to measure the efficiency of editing by Western Blot. Furthermore, cell survival experiment (MTT assay) was employed to measure the effect of PLAC8 knockout on cell proliferation. Meanwhile, the expression levels of the proliferation-related genes in PLAC8 knockout 293T cells were detected via Western Blot. In conclusion， we successfully constructed PLAC8-knockout cell line. Compared with wildtype 293T, knockout of PLAC8 could inhibit cell proliferation. The decreased cell proliferation was associated with the reduced expression of AKT and C-MYC and increased expression of RAF-1 and ERK2, which indicated that PLAC8 might regulate cell proliferation via AKT and RAF-1-ERK2-C-MYC cascade signal pathway

Key words: CRISPR/Cas9, knockout, PLAC8, cell proliferation, MAPK signal pathway